Mx3000p

Two‐week‐old ROX‐Myc^OsERF48 and NT plants grown on soil were hydroponically adapted in water for 3 days and then fixed by cross‐linking with 1% formaldehyde under vacuum for 15 min. Cross‐linking was stopped by the addition of glycine to a final concentration of 125 mm and application of vacuum for 10 min. After washing the plants in cold water, roots were collected and frozen in liquid nitrogen, and stored at −80 °C. The ChIP assay was performed as described by Chung et al. (2009), except that an anti‐myc antibody (SC‐789; Santa Cruz Biotech, Santa Cruz, CA) used. The ChIP product was analysed via quantitative PCR on a Mx3000P real‐time PCR system (Agilent Technologies). The enrichment values were normalized to the input sample. Values are the means ± SD of three independent experiments. All primer sequences are listed in Table S3.

For the gene expression analysis in leaf blades, total RNA was isolated from two 1-cm long leaf sections per plant spotted with a conidial suspension. For the analysis in leaf sheaths, total RNA was isolated from two 1.5-cm long sections of inoculated leaf sheaths per plant. Total RNA was extracted using Sepasol RNA I Super (Nacalai Tesque, Kyoto, Japan). First strand cDNA was synthesized using the PrimeScript RT reagent kit (Takara Bio, Kusatsu, Japan). qRT-PCR was performed using SYBR Premix Ex Taq II (Takara Bio), and the relative levels of gene expression were quantified using MX3000P (Agilent Technologies Inc., Santa Clara, CA, USA). Data were normalized to the expression levels of eEF-1α in rice and ACT1 in M. oryzae. Primer sequences are listed in S4 Table.

BMDMs were stimulated using LPS. Total RNA was extracted using a Trizol reagent (Takara, Japan), and reverse-transcribed into cDNA using a cDNA Synthesis Kit (Takara, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using an Agilent Mx3000P machine and the TransStart Green qPCR SuperMix (Takara, Japan), according to the manufacturer's instructions. Each sample was conducted in triplicate and the gene expression levels were calculated relative to the amount of GAPDH using the 2^−ΔΔCT method. The primer sequences for the tested genes were listed in Table 1.

The expression of the genes was carried out by real-time PCR (RT-qPCR). To do this, a Stratagene Mx3000P thermal cycler was used (Agilent Technologies, United States). The amplification of the cDNA was done using the Maxima SYBR Green qPCR Master Mix (2X) kit (Thermo Scientific) in a final volume of 20 μl, containing 50 ng (2 μl) of cDNA, 10 μl of Maxima SYBR Green qPCR Master Mix (2X), 10 nM of forward and reverse for each gene, completing the final volume with nuclease-free water. For each biological replication, two technical replicas were made. The amplification profile used the following temperature: 95°C for 10 min, 40 cycles of 95°C × 30″, 60°C × 1′, 72°C × 20″ (Vashisth et al., 2011 (link)). A dissociation curve was generated at the end of each amplification program to verify that a single product was amplified, for which the temperature was increased from 55 to 95°C with a continuous fluorescence measurement. The calculations of the relative expression of each gene were made according to Pfaffl (2001) (link) and analyses were made in relation to the normalizing gene GAPDH (Czechowski et al., 2005 (link)). The relative expression was recorded in 12 individuals per treatment and along a temporal kinetic at 0, 12, 36, 48, and 120 h.